Materials and Methods: A batch of newly hatched hybrid grouper fry (Epinephelus fuscoguttatus × Epinephelus lanceolatus) were followed from the larval stage to market size. Samples of the hybrid groupers, water, live feed, and artificial fish pellets were collected periodically from day 0 to 180 in the hybrid grouper hatchery. Reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR amplifications were carried out on VNN-related sequences. The phylogenetic tree including the sampled causative agent of VNN was inferred from the coat protein genes from all known Betanodavirus species using Molecular Evolutionary Genetics Analysis (MEGA). Pearson's correlation coefficient values were calculated to determine the strength of the correlation between the presence of VNN in hybrid grouper samples and its associated risk factors.
Results: A total of 113 out of 146 pooled and individual samples, including hybrid grouper, water, and artificial fish pellet samples, demonstrated positive results in tests for the presence of VNN-associated viruses. The clinical signs of infection observed in the samples included darkened skin, deformation of the backbone, abdominal distension, skin lesions, and fin erosion. VNN was present throughout the life stages of the hybrid groupers, with the first detection occurring at day 10. VNN-associated risk factors included water temperature, dissolved oxygen content, salinity, ammonia level, fish size (adults more at risk than younger stages), and life stage (age). Detection of VNN-associated viruses in water samples demonstrated evidence of horizontal transmission of the disease. All the nucleotide sequences found in this study had high nucleotide identities of 88% to 100% to each other, striped jack nervous necrosis virus (SJNNV), and the reassortant strain red-spotted grouper NNV/SJNNV (RGNNV/SJNNV) isolate 430.2004 (GenBank accession number JN189932.1) (n=26). The phylogenetic analysis showed that quasispecies was present in each VNN-causing virus-positive sample, which differed based on the type of sample and life stage.
Conclusion: This study was the first to confirm the existence of a reassortant strain (RGNNV/SJNNV) in hybrid groupers from Malaysia and Southeast Asia. However, the association between the mode of transmission and the risk factors of this virus needs to be investigated further to understand the evolution and potential new host species of the reassortant strain.
MATERIALS AND METHODS: Seven broodstock pairs of P. scalare were used in this study to follow the life stages of fish, from egg to market size. Water samples and other samples, such as mucus swabs, gill swabs, P. scalare eggs, fries, juveniles, snails, snail eggs, live feed (Tubifex worms and Moina spp.), sediment samples, and wild fish, were collected periodically for initial environmental sampling from day 0 to day 60. Nested polymerase chain reaction amplifications were performed for megalocytivirus-related sequences. The phylogenetic tree, including the sampled causative agents of megalocytiviruses, was inferred from the major capsid protein genes of all known Iridoviridae species. Pearson's correlation coefficients were calculated to determine the strength of the correlation between the presence of megalocytiviruses in P. scalare samples and the associated risk factors.
RESULTS: A total of 312 out of 935 pooled and individual samples tested positive for the presence of Megalocytivirus-related sequences, except snail eggs and wild fish (Poecilia reticulata). No clinical symptoms were observed in any fish samples. Megalocytivirus-associated viruses detected in water samples indicate horizontal transmission of the virus. All the nucleotide sequences found in this study had high nucleotide identities of 95%-99 % and were closely related to Megalocytivirus genotype I infectious spleen and kidney necrosis virus. Risk factors associated with Megalocytivirus include water temperature, dissolved oxygen (DO), pH, ammonia, nitrate, nitrite, and the life stages of P. scalare. High Megalocytivirus infection was detected when the water temperature, DO, and pH were high in P. scalare, high water temperature and nitrate in the water samples, and the same rate of Megalocytivirus infection in P. scalare fry and juveniles.
CONCLUSION: This is the first study to confirm the existence of different possible routes of megalocytivirus distribution in ornamental fish farms in Malaysia. Nevertheless, the connection between the mode of transmission and the risk factors for this virus needs to be explored further to recognize the evolution and potential new host species.